Abstract

Three treatments: S0, S0.4 (0.4 %
added sulphur) and S0.8 (0.8 % added sulphur) were compared in an in vitro
incubation with 3 repetitions in a completely randomized design, using
as substrate a basal diet of NaOH-treated rice straw, molasses and cottonseed
meal. The forage components of the diets were dried and milled through a 1 mm
screen and mixed with the other components of the diet. Representative samples
of the substrate (20 g DM) were put in an incubation flask (2500 ml) to which
were added 1.6 liters of buffer solution and 400ml of rumen fluid. The rumen
fluid for each treatment was obtained from rumen-fistulated cattle that were on
the same dietary treatments. The flasks were then incubated at 380C
in a water bath for 72h. During the incubation, each flask was connected to an
aluminium bag for total collection of gas over the 72h incubation. At the end of
the incubation the total gas volume was recorded and samples analyzed for the proportion of methane. The same treatments and
the same methods were repeated to measure the loss of substrate during an
incubation of 24h.

Methane production per unit substrate fermented
was reduced linearly with increase in level of sulphur in the incubation medium.

Introduction

This paper is part of a series of researches aimed at optimizing the use of
nitrate salts as a strategy for reducing methane emissions from enteric
fermentation in ruminants in order to meet future targets for mitigating global
warming (Leng 2008). Trinh Phuc Hao et al (2009) and Le Thi Ngoc Huyen et al
(2010) showed that nitrate could be safely fed as the major source of
fermentable N provided that the animals (goats and cattle, respectively) were
adapted to the diet over a period of 2 weeks. The positive effect of nitrate
salts in reducing methane production has been shown in both in vitro (Binh
Phuong et al 2011; Inthapanya et al 2011; Outhen et al 2011; Thanh et al 2011)
and in vivo (Anh Nguyen Ngoc et al
2010; Van Zijderveld et al 2010)The study of Sokolowski et al (1969)
indicated that there was an apparent interaction between nitrate and sulphate
with N retention and wool growth being enhanced when both were present in the
diet, compared with either nitrate or sulphate given separately. The results
reported by Van
Zijderveld et al (2010) and Silivong et al (2011) also showed a
synergistic effect on methane mitigation of feeding both nitrate and sulphate.

The objective of the present research was to study the effects of nitrate and
sulphur on methane production and DM fermentation in an in vitro system
using rice straw and molasses as the major substrates.

Materials and methods

Location and experiment design

The experiment was
conducted in the Laboratory of the Department of Animal Science, College
of Agriculture and Applied Biology, Can Tho Uinversity.

Treatments and design

The treatments in a completely randomized design with three
repetitions were:

S0: Basal diet with no
added sulphur

S0.4: Basal diet with
addition of 0.4% sulphur

S0.8: Basal diet with
addition of 0.8% sulphur

The sulphur was in the form of the pure element. Sodium nitrate
was included in all diets as the source of NPN (Table 1).

Table 1.
Ingredients of experiment
diets (%DM)

Feeds

T1-SN

T2-SD-0.4%-S

T3-SD-0.8%-S

NaOH-Rice
straw

48.4

48.4

47.6

Molasses

20

20

20

Cotton seed
meal

20

20

20

Grass

5

5

5

Sodium
nitrate

6.6

6.6

6.6

Sulphur (S)

0

0.4

0.8

Preparation of substrates for the in vitro incubation

Rice straw was chopped to lengths of 3-4cm. A solution was prepared containing
3% NaOH and 97% water. This solution was then sprayed on the rice straw at the
rate of 30 litres solution to 100 g rice straw DM. The treated rice
straw, grass and cottonseed meal were dried at 60°C and milled through a 1 mm
screen. A representative sample (20 g DM ) was put in
an incubation flask (2500ml) to which were added 1.6 liters of buffer solution
and 400ml of rumen fluid, prior to filling each flask with carbon dioxide. A
separate incubation was carried out with the same substrates to determine DM
fermented over a 24h period. For this study, a representative sample of the
substrates (0.5 g DM ) was put in an incubation flask (100ml) to which were
added 40 ml of buffer solution and 10 ml of rumen fluid, prior to filling each
flask with carbon dioxide. The rumen fluid for each treatment was obtained from
rumen-fistulated cattle that were on the same dietary treatments. The flasks
were incubated at 38°C in a water bath for 72h for measuring methane
and for 24h for measuring DM degradation. During the incubation for methane
production, each flask was connected to an aluminium bag for total collection of
gas over the 72h period. At the end of the incubation the total gas volume was
recorded and samples analyzed for the proportions of
methane and carbon dioxide.

Measurements

Total gas volume was recorded with a "Ritter" gas flow meter (Calibrated
Instrument Inc; Photo 1) and
samples
analyzed for the percentage of methane using a gas
detector (Geotechnical Instruments GA 94; Photo 2). Feed samples were analyzed for DM, ash, NDF,
ADF and N according to AOAC (1990).

Photo 1. The gas flow meter

Photo 2. The gas meter for measuring methane

Statistical analysis

The results were analyzed by the General Linear Model option in the ANOVA
program of the Minitab Software (version13.2). Sources of variation in the model
were: level of added sulphur and error.

Results and Discussion

The chemical composition of the ingredients in the substrate is shown in Table
2.

Due to error, the measurement of substrate fermented was only determined after
24h of incubation, while gas measurements were made after 72h. There appear to
be no data in this type of in vitro system for effect of incubation time
on proportion of substrate fermented at times varying from 4h to 72h. However,
in a recent report Inthapanya et al (2010) plotted substrate fermented against
time over the period 4 to 24h using a substrate containing 67% alkali-treated
rice straw and 30% cassava leaves (DM basis), with the NPN source being
potassium nitrate or urea. The relationship of DM fermented with incubation time
over the 24h period was linear with no difference between the slopes for urea
and potassium nitrate. In the present experiment, the values for methane
production per unit substrate fermented (calculated as methane production at 72h
and substrate fermented at 20h) will not be the same as if both had been
determined at the same incubation time (either 24 or 72h); however, it appears
reasonable to assume that the relative differences due to source of additive (in
this case sulphur) would be similar.

There was a tendency (P=0.18) for the methane content of the gas after 72
h to be lower for the treatments with added sulphur (Table 3). The proportion of
the substrate fermented after 24h was increased due to supplementation with
sulphur. Methane production (at 72h) per unit of substrate fermented (at 24h)
decreased linearly with sulphur supplementation (Figure 1).

Table 3.
Mean values for methane content of the gas at 72h, substrate
fermented after 24h and the production of methane (at 72h) per unit
DM of substrate fermented (at 24h)

Sulphur added, % in DM

0

0.4S

0.8S

SEM

P

#CH4 in the gas, %

18.0

17.5

17.6

0.16

0.18

β Substrate DM fermented , %

32.1b

35.0a

36.5a

0.464

0.027

#,β CH4, ml/g DM
fermented

7.67a

7.41a

6.82b

0.136

0.006

# Methane production after 72h ;β Substrate fermented after 24hab Means without common superscript are different at P<0.05

Figure 2. Relationship between sulphur level and methane production at 72h per unit substrate fermented after 24h

The
reduction in methane production with added sulphur is in agreement with the
findings of Van Zijderveld et al (2010), Silivong et al (2011) and Binh Phuong
et al (2011). The results of Binh Phuong et al (2011) are of interest as they
indicate that the synergism between the NPN and sulphur on methane mitigation
occurred only when the NPN source was nitrate and not urea.

Conclusions

Methane production per unit substrate fermented
was reduced linearly with increase in level of sulphur in an incubation medium
that contained sodium nitrate.

Binh Phuong L T, Preston T R and Leng R A 2011
Mitigating methane production from ruminants; effect of supplementary sulphate
and nitrate on methane production in an in vitro incubation using sugar cane
stalk and cassava leaf meal as substrate. Livestock Research for Rural
Development. Volume 23, Article #22.
http://www.lrrd.org/lrrd23/2/phuo23022.htm

Inthapanya S, Preston T R and Leng R A 2011 Mitigating methane production from ruminants; effect of calcium nitrate as
modifier of the fermentation in an in vitro incubation using cassava root as the
energy source and leaves of cassava or Mimosa pigra as source of protein.
Livestock Research for Rural Development. Volume 23, Article #21.
http://www.lrrd.org/lrrd23/2/sang23021.htm

Thanh V D, Preston T R and Leng R A 2011
Effect on methane production of supplementing a basal substrate of
molasses and cassava leaf meal with mangosteen peel (Garcinia mangostana) and
urea or nitrate in an in vitro incubation. Livestock Research for Rural
Development. Volume 23, Article #98.
http://www.lrrd.org/lrrd23/4/than23098.htm

Tilley J M A and Terry R A
1963 A two stage technique for the in vitro digestion of forage crops.
Journal of the British Grassland Society 18 : 104.